Phosphorylation of PABPN1 during mitosis protects mRNA from hyperadenylation and maintains transcriptome dynamics.. Phosphorylation of PABPN1 during mitosis protects mRNA from hyperadenylation and maintains transcriptome dynamics.

Polyadenylation controls mRNA biogenesis, nuclear export, translation, and decay. These processes are interdependent and coordinately regulated by poly(A)-binding proteins (PABPs), yet how PABPs are themselves regulated is not fully understood. Here, we show that human PABPN1, the nuclear PABP, is phosphorylated by mitotic kinases at four specific sites during mitosis, a time when nucleoplasm and cytoplasm mix. Phospho-inhibitory mutations decreased human cell proliferation. We therefore employed long-read sequencing to determine how PABPN1 phospho-site mutants affected poly(A) tails lengths of individual mRNAs, and TimeLapse-seq to monitor mRNA synthesis and decay. Phospho-inhibitory PABPN1 mutants lengthened poly(A) tails on both spliced and unspliced transcripts. In contrast, expression of phospho-mimetic PABPN1 resulted in shorter poly(A) tails and reduced transcriptome stability, indicating reduced polyadenylation activity of PABPN1 and targeting of long-tailed transcripts for decay. Thus, PABPN1 phosphorylation confers transcriptome instability that is associated with resetting the gene expression program as cells passage through cell cycle. Overall design: To investigate the role of PABPN1 phosphorylation in mRNA processing, we constructed HEK293T cell lines with Doxycycline-inducible expression of WT PABPN1, 4SA PABPN1 (S19, S150, S197, and S304 to A), or 4SD PABPN1 (S19, S150, S197, and S304 to D).

多聚腺苷酸化（polyadenylation）调控mRNA生物发生、核输出、翻译与降解。这些过程相互依存，并由多聚A结合蛋白（poly(A)-binding proteins, PABPs）协同调控，但目前对于PABPs自身的调控机制尚未完全阐明。本研究发现，人类核型多聚A结合蛋白PABPN1在有丝分裂期会被有丝分裂激酶在四个特定位点发生磷酸化，而有丝分裂期间核质与细胞质会发生混合。磷酸化抑制型突变会削弱人类细胞增殖能力。为此，我们采用长读长测序（long-read sequencing）分析PABPN1磷酸化位点突变体对单个mRNA的poly(A)尾长度的影响，并借助TimeLapse-seq技术监测mRNA的合成与降解过程。实验结果表明，磷酸化抑制型PABPN1突变体可延长剪接型与非剪接型转录本的poly(A)尾长度；与之相反，表达磷酸化模拟型PABPN1则会缩短poly(A)尾长度并降低转录组稳定性，这提示PABPN1的多聚腺苷酸化活性被抑制，且长尾转录本会被靶向降解。综上，PABPN1磷酸化可引发转录组不稳定性，这与细胞周期进程中基因表达程序的重置密切相关。

总体实验设计：为探究PABPN1磷酸化在mRNA加工中的功能，我们构建了可经强力霉素诱导表达野生型（WT）PABPN1、4SA型PABPN1（将第19、150、197、304位丝氨酸突变为丙氨酸）或4SD型PABPN1（将第19、150、197、304位丝氨酸突变为天冬氨酸）的HEK293T细胞系。